Single-cell transcriptomes and T cell receptors of vaccine-expanded apolipoprotein B-specific T cells

Abstract

Atherosclerotic cardiovascular diseases are the major cause of death worldwide. CD4 T cells responding to Apolipoprotein B (ApoB), the core protein of most lipoproteins, have been identified as critical disease modulators. In healthy individuals, ApoB-reactive (ApoB⁺) CD4 T cells are mostly regulatory T cells (T_regs), which exert anti-inflammatory effects. Yet, they may obtain pro-inflammatory features and thus become proatherogenic. Evidence from animal studies suggests that vaccination against certain major histocompatibility complex (MHC) II-binding ApoB peptides induces an expansion of ApoB⁺ T_regs and thus confers atheroprotection. To date, in-depth phenotyping of vaccine-expanded ApoB⁺ T cells has not yet been performed. To this end, we vaccinated C57BL/6J mice with the ApoB-peptide P6 (ApoB_978-993 TGAYSNASSTESASY) and performed single-cell RNA sequencing of tetramer-sorted P6⁺ T cells. P6⁺ cells were clonally expanded (one major, two minor clones) and formed a transcriptional cluster distinct from clusters mainly containing non-expanded P6⁺ and P6^- cells. Transcriptomic profiling revealed that most expanded P6⁺ cells had a strong T_reg signature and highly expressed genes mediating suppressive functions. Yet, some expanded P6⁺ cells only had a residual T_reg signature and expressed genes related to T helper 1 (T_H1) cells, which are proatherogenic. Modeling the T cell receptor (TCR) and P6:MHC-II interaction showed that only three amino acid residues in the α and β chain contact the P6 peptide in the MHC-II groove and thus determine the specificity of this TCR to P6. Our data begin to reveal the vaccination-induced response to an ApoB epitope.

Keywords: ApoB; P6; atherosclerosis vaccine; regulatory T cells; single-cell RNA-sequencing.

FIGURE 1

Common TCR α and β chains among the P6⁺ and P6^– cells. Each circle represents a cell. The expanded P6⁺ cells (orange, green and blue circle) have at least one chain in common amongst themselves. A few non-expanded P6⁺ cells also have some common chains with expanded P6⁺ cells even though they may not belong to the same clonotype. Expectedly, P6^– cells mostly do not have chains in common. Lines represent at least one common chain between the cells at its termini. Line color represents which chain (s) is shared. Clonotypes were based on having the exact same reconstructed chains for both productive and non-productive chains. Cells with no successful reconstructed chains were not included in this figure.

FIGURE 2

Productive α and β chain reconstruction for the P6⁺ cells. (A) Pie charts depicting the frequency of each gene for V and J segment in both productive chains among tetramer⁺ cells. (B) Table listing the reconstructed genes for each productive chain among P6⁺ cells as well as the size of the clonotype. Blank fields represent cases in which the reconstruction algorithm failed to reconstruct a chain. Two unexpanded cells were excluded because no productive chains were identified. Clonotype calling was based on both productive and non-productive chains.

FIGURE 3

Clustering based on transcriptional profiles correlates with clonality. (A) UMAP projection of the cells. This projection is based on the first 8 principal components of the normalized imputed expression data of the highly variable genes. Graph-based clustering revealed three major clusters (orange, green, blue). (B) The same UMAP projection but with cells labeled according to their clonotypes. Expanded P6⁺ cells are separated into three clonotypes (orange, green, blue) based on their productive and non-productive TCRα and TCRβ chains. Expanded group 3 (blue) cells are separated from the remainder of the expanded P6⁺ cells. Undetermined cells are cells for which TCR information is unavailable.

FIGURE 4

Hierarchical clustering of the normalized expression heatmap of 38 lineage-defining genes across all P6⁺ cells. Columns were clustered according to Ward’s second clustering criterion. Four segments were automatically generated based on the hierarchical clustering. All clusters with expanded P6⁺ cells (clusters 1, 2, 3) show evidence of a T_reg signature.

FIGURE 5

I-A^b ApoB P6 peptide and TCR modeling. (A) Overview of the TCR/I-A^b/ApoB P6 complex with I-A^b in gray, TCRα in orange and TCRβ in green. ApoB P6 peptide in yellow sticks. (B) Binding of the ApoB P6 peptide in the I-A^b binding groove identifies amino acid residues that are accessible for TCR binding. (C) TCR-ApoB P6 contact residues. (D) TCRα contact residues highlighted orange and TCRβ residues green. The ApoB P6 core peptide is underlined and contact residues are highlighted yellow.